Mimicking Nonribosomal Peptides from the Marine Actinomycete Streptomyces sp. H-KF8 Leads to Antimicrobial Peptides

Microorganisms within the marine environment have been shown to be very effective sources of naturally produced antimicrobial peptides (AMPs). Several nonribosomal peptides were identified based on genome mining predictions of Streptomyces sp. H-KF8, a marine Actinomycetota isolated from a remote Northern Chilean Patagonian fjord. Based on these predictions, a series of eight peptides, including cyclic peptides, were designed and chemically synthesized. Six of these peptides showed antimicrobial activity. Mode of action studies suggest that two of these peptides potentially act on the cell membrane via a novel mechanism allowing the passage of small ions, resulting in the dissipation of the membrane potential. This study shows that though structurally similar peptides, determined by NMR spectroscopy, the incorporation of small sequence mutations results in a dramatic influence on their bioactivity including mode of action. The qualified hit sequence can serve as a basis for more potent AMPs in future studies.


Amino acid analysis
The method for the quantification of individual amino acids within the synthesized linear peptides (L1, L2 and L3) is based on a procedure optimized from previous publication (Baumruck et all 2021). 79Quantification was done by complete peptide hydrolysis, subsequent ACQ derivation followed by HPLC separation and UV quantification of the individual amino acid signals.External calibration for the quantification was done by measuring peak areas of dilutions of a known amino acid standard.All HPLC injections were done in replicates in order to demonstrate reproducibility and the mean values of both measurements were processed for calculations.
HPLC analysis was performed using MultoHigh 100 RP18 3μ column (125 x 4 mm) at a column temperature of 45°C.Eluent A was sodium acetate buffer (50 mM, pH 5.75) and eluent B was 70% acetonitrile, 30% sodium acetate buffer (50mM, pH 6.0).The gradient method started with an isocratic mixture of 2 % eluent B for 2.5 min followed by a linear gradient of 2 -30 % eluent B over 70 min and short final linear gradient of 30 -70 % eluent B for 5 min at a flow rate of 1 mL/min.Chromatograms were extracted and analyzed at 254 nm.
HPLC for linear calibration

Calculation of peptide content
Table S3.Amino acid analysis: HPLC peak areas used for the calculation of the peptide content.With the slope, intercept and the average of two measured peak areas at 254 nm the average amino acid concentration (avgCx) for each specific amino acid was calculated.The following equation leads to the amino acid content:

Amino acid
(%) =  *  *  . (..)*      Please note, that ehe average amino acid concentration (avgCx) is multiplied by a factor of 2, because 10 μl of the hydrolyzed and derivatized peptide samples were injected instead of 20 μl as used in the amino acid calibrations.For C2 NH chemical shifts at 275K

NMR
(2) For C2 NH chemical shifts at 283K (3) For C2 NH chemical shifts at 290K For C2 NH chemical shifts at 298K

Minimum microbicidal concentration (MMC), killing 99% of the inoculum.
All microorganisms were cultured in brain-heart infusion broth (BHI) overnight on a shaker at 37°C.The cultures were thereafter transferred to fresh BHI broth in volumes constituting 10% of total, and incubated for an additional 2 h to reach exponential growth phase.The bacterial cultures were washed once in BHI diluted 1/100 (BHI 100 ), followed by resuspension in BHI 100 .The cell suspensions were diluted to a density of 1x10 7 (bacteria) or 2x10 7 CFU/ml (yeast), as estimated by optical density measurement at 590 nm.
Peptides were serially diluted by twofold steps in a microtiter plate (Sarstedt, Numbrecht, Germany, 82.1581001) in duplicate or triplicate (200 µl per well), starting with 100 µg/ml as the highest concentration unless otherwise stated.The diluents used were BHI 100 , and BHI 100 with 85 or 150 mM NaCl.The bacterial or yeast cell suspensions were added in 10-µl volumes to the wells, giving a final concentration of approximately 5 x 10 5 cells/ml.The initial concentration of the inoculum was checked by viable counts.The microplate was incubated at 37°C in a humid chamber for 24 h.Viability of the inoculum was analysed at 2, 6 and 24 h by culturing 5 µL of the suspension of each well, added as a drop onto blood agar plates (blood agar supplemented with 5% defibrinated horse blood).After incubation overnight at 37 •C, the viable count in each drop was recorded, and the concentration of CFU/ml calculated.The MMC of a peptide causing ≥99% reduction of the inoculum was defined as MMC 99 .
Figure S3.(I) RP-HPLC chromatogram (gradient from 10 to 50% acetonitrile in water over 10 min at 2 ml/min, detection at 214 nm) and (II) high resolution mass spectrum for peptide L1.

Figure S11 .
Figure S11.Calibration curves of the amino acids T, A and D used for the peptide content calculation.

Figure S12 .
Figure S12.CD spectra of linear synthesized peptides Lin1 in red, Lin2 in green and Lin3 in blue in two different solvents and concentration.A) 10µM in water, 293 K, b) 50µM in water, 293 K.

Figure S 16 .
Figure S 16. Cell length measurements of E. coli CCUG31246 after 10 and 60 min treatment with C3 L3, L3-K, and PolB.Black lines indicate the median of each sample.Cells from three independent biological replicates were pooled for analysis.A minimum of 91 cells was analyzed per sample.
*Based on BLASTp results

Table S2 .
Analytical data for synthesized peptides.

Table S5 .
Energy and structural ensembles of synthesized peptides.

Table S6 .
Antimicrobial activity of L2 and L3 in the presence of NaCl. 1 L2 and L3 were serially diluted in twofold steps with a starting dilution of 200 µg/ml except for L3 in 150 mM NaCl, where 400 µg/mL were used for the bacterial strains.All sample dilutions were run in duplicates (L2) or triplicates (L3).2Thegrowth of S. aureus at 24 h in BHI 100 with 85 or 150 mM NaCl was reduced.